Iron binding substances have been identified within the microvilli and apical cytoplasm of the mucosal absorptive cells in the duodenum and jejunum of rats and humans. Ultrastructural and immunologic studies suggest that these substances are neither transferrin, transferrin receptors nor ferritin. The observation that they avidly bind other divalent metal cations in vivo demonstrates a biochemical function which differentiates them from the known iron binding proteins in the intestinal mucosal cells and suggests that they are participants in a shared absorptive pathway for divalent metals which may explain why iron and cobalt are absorbed competitively, iron deficient subjects are more susceptible to lead poisoning, and calcium inhibits the absorption of iron. Preliminary studies suggest that this putative protein has a large molecular size (370000 daltons) which makes it difficult to separate from ferritin (460000 daltons) in mucosal homogenates. We propose to utilize mucosal homogenates as an initial step in separation of this large protein. Using chromatographic methods, we plan to isolate this metal binding substance so that it can be characterized. In addition, once sufficient quantities are isolated, we plan to raise monoclonal antibodies which then can be used to obtain larger yields of the protein and as a probe to measure the quantities of this substance in the intestinal mucosa of animals in various states in which iron absorption is affected to determine if quantitative changes in the amount of this protein occur to regulate the absorption of iron and other metals. Similar isolates of vitamin D calcium binding protein will be accomplished from mucosal homogenates because this also seems to bind the devalent metal cations competitively. The quantity of the new large putative metal binding protein, ferritin, mucosal transferrin and vitamin D dependent calcium binding protein will be measured in subcellular fractions of intestinal mucosal cells from animals in the various states of iron repletion. Similar studies will be performed in other body organs to include placenta. We postulate that this information will add significantly to an understanding of factors regulating iron absorption.